This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
Article Cite This: ACS Omega 2019, 4, 5540−5546
http://pubs.acs.org/journal/acsodf
Expression, Purification, and Characterization of Human Diacylglycerol Kinase ζ Takumi Saito,† Daisuke Takahashi,‡ and Fumio Sakane*,† †
Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan Department of Pharmaceutical Health Care and Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
‡
Downloaded via 46.148.127.28 on March 26, 2019 at 00:20:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: Diacylglycerol kinase ζ (DGKζ) phosphorylates diacylglycerol (DG) to generate phosphatidic acid. The dysfunction of DGKζ has been linked to several diseases, such as cardiac hypertrophy, ischemia, and seizures. Moreover, much attention has been paid to DGKζ, together with DGKα, as a potential target for cancer immunotherapy. However, DGKζ has never been purified and, thus, neither its enzymatic properties nor its structure has yet been reported, hindering our understanding of the catalytic mechanism of DGKζ and the development of a reasonable structure-based drug design. In the present study, we generated a full-length DGKζ using a baculovirus−insect cell expression system for enzymological and structural studies. Full-length DGKζ remained soluble and was purified to near homogeneity as a monomer with yields suitable for protein crystallization (0.63 mg/1 L culture). Enzymatic characterization showed that the purified DGKζ is in a fully functional state. The Km values for adenosine triphosphate (ATP) and DG were 0.05 mM and 1.5 mol %, respectively, and the EC50 for the activator phosphatidylserine was 8.6 mol %, indicating that its affinity for ATP is moderately higher than those of DGKα and DGKε, and its affinities for DG and phosphatidylserine are comparable to those of DGKα/DGKε. We further confirmed that the purified enzyme could be concentrated without any significant aggregation. Circular dichroism revealed that DGKζ is comprised of 25% α-helices and 18% β-strands. This is the first successful purification and characterization of the enzymatic and conformational properties of DGKζ. The purification of DGKζ allows detailed analyses of this important enzyme and will advance our understanding of DGKζ-related diseases and therapies.
■
attenuated cell proliferation.11 Liu et al. reported that DGKζ constitutes a downstream component of the leptin signaling pathway in the hypothalamus.12 In NIE-115 neuroblastoma cells, DGKζ promotes neurite outgrowth.13 We previously showed that retinoic acid-induced neurite outgrowth was attenuated by a deficiency of DGKζ, which produced 16:0/ 16:0-PA species in Neuro-2a neuroblastoma cells.14 In that study, we observed the role of DGKζ in the morphological changes at the initial/early stages of neuronal differentiation. Rac1 is essential for retinoic acid-induced neurite extension of Neuro-2a cells.15 DGKζ-derived PA activates p21-activated protein kinase 1, which induces the release of Rac1 from Rho guanine nucleotide dissociation inhibitors.16 DGKζ-mediated synaptic conversion of DG to PA is involved in the maintenance of dendritic spines.17 In addition, DGKζ attenuates the hypertrophic signaling cascade and resultant cardiac hypertrophy in response to Gq protein-coupled receptor agonists.18 DGKζ in hippocampal neurons is involved in global ischemia19 and kainate-induced seizures.20 Moreover,
INTRODUCTION Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA).1−4 DG is well known to be an activator of conventional and novel protein kinase C (PKC), Ras guanyl nucleotide-releasing protein, chimaerins, and Unc-13.5−7 Moreover, PA has been reported to regulate the activities of many physiologically important enzymes such as Ras GTPase-activating protein, phosphatidylinositol-4-phosphate kinase, Raf-1 (C-Raf) kinase, atypical PKC, and mammalian target of Rapamycin.8,9 Thus, DGK appears to participate in a wide variety of physiological and pathological events by controlling the balance between two lipid second messengers, DG and PA. To date, 10 mammalian DGK isozymes (α, β, γ, δ, ε, ζ, η, θ, ι, and κ) have been identified. These DGK isozymes are divided into five groups (type I (α, β, and γ), II (δ, η, and κ), III (ε), IV (ζ and ι), and V (θ)) according to their structural features.1−4 Type IV DGK isozymes (ζ and ι) commonly contain two C1 domains at the N-terminus, a MARCKS-like domain, four ankyrin repeats, and a PDZ domain at the Cterminus in addition to a catalytic domain (CD).10 DGKζ is a versatile enzyme. Topham et al. revealed that reducing nuclear DG levels through the action of DGKζ © 2019 American Chemical Society
Received: January 9, 2019 Accepted: March 8, 2019 Published: March 19, 2019 5540
DOI: 10.1021/acsomega.9b00079 ACS Omega 2019, 4, 5540−5546
ACS Omega
Article
Figure 1. Expression of DGKζ-FL in baculovirus-infected insect cells and purification. (A) Schematic domain architecture of DGKζ-FL. (B) Immunoblot analysis of the solubility of DGKζ-FL expressed in Sf9 cells. Cell lysates were separated into a 25 000g supernatant (Sup) and pellets (Ppt) and subjected to SDS-PAGE (7.5%) followed by immunoblot analysis using anti-DGKζ antibody. (C) SDS-PAGE (7.5%) analysis of fractions from Ni2+-affinity purification; separated proteins were stained with Coomassie blue staining. (D) Elution profile of DGKζ-FL from size exclusion chromatography; fraction numbers (elution volume) used for the following SDS-PAGE analysis are labeled. Inset shows the calibration of the gel-filtration column using protein standards of known molecular weight (thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa)). The partition coefficient (Kav) was calculated from the formula Kav = (VE − V0)/(VT − V0), where VE is the retention volume of each sample, VT is the total column volume (120 mL), and V0 is the void volume of the column (44 mL). Kav was plotted against the molecular weight of the proteins, and linear regression analysis was conducted. (E) SDS-PAGE (7.5%) analysis of DGKζ-FL purified using sizeexclusion chromatography.
we now recognize the importance of DGKζ as a physiological negative regulator of T-cell receptor signaling and T-cell activation. DGKζ-deficient T cells were hyperresponsive to Tcell receptor stimulation both ex vivo and in vivo.21,22 Therefore, it is thought that selective inhibitors of DGKζ enhance T-cell receptor signaling and, consequently, can be promising anticancer drugs through cancer immunity.23−26
However, despite their physiological and biomedical importance, no structural and enzymatic properties of DGKζ have been determined, hindering our understanding of the catalytic mechanism of DGKζ and the development of a reasonable structure-based medicine design. In the present study, a full-length human DGKζ expressed in the soluble form in the baculovirus−insect cell expression system was 5541
DOI: 10.1021/acsomega.9b00079 ACS Omega 2019, 4, 5540−5546
ACS Omega
Article
exist in two alternatively spliced forms.10,32 We selected human DGKζ1,10 which is ubiquitously expressed.10,32 The catalytic domain is an essential target for inhibitor development. Thus, we first attempted to express human DGKζ-CD (aa 276−629) in Sf9 cells. The cDNA construct of DGKζ-CD (Figure S1A) with an N-terminal His × 6 tag was expressed in Sf9 insect cells and purified using Ni-NTA affinity chromatography (Figure S1B). However, DGKζ-CD was not highly soluble (64% soluble) (Figure S1B) and was recovered in flow-through fractions in Ni-NTA affinity chromatography. Therefore, the amount of Ni-NTA-purified DGKζ-CD was low (85 μg/L of Sf9 cell culture) (Figure S1C). We previously reported that the full-length DGKα expressed in Sf9 cells was soluble.27 We therefore tried to produce human DGKζ-FL (aa 1−928) (Figure 1A) using the baculovirus expression system. Western blot analysis showed that 96% of DGKζ-FL was soluble after cell lysis (Figure 1B). DGKζ was purified using Ni-affinity chromatography from the cell lysis supernatant and eluted in the fractions containing 50 and 100 mM imidazole (Figure 1C). Next, to further purify DGKζ-FL, size-exclusion chromatography on a Superdex 200 column was performed after concentration. DGKζ-FL was eluted as a single peak at the molecular mass of 125 kDa based on a calibration curve obtained with molecular mass standard proteins (Figure 1D). The calculated molecular mass of His × 6-tagged DGKζFL is 106 kDa (DGKζ (∼104 kDa) + His × 6 tag (∼2 kDa)).10,33 Therefore, it is likely that DGKζ-FL exists as a monomer in solution. Overall, DGKζ-FL was purified to near homogeneity (Figure 1E), and the yield was ∼0.63 mg per 1 L of Sf9 cell culture. Enzymatic Properties of Purified DGKζ. To examine whether the purified DGKζ-FL is catalytically active, we performed the octyl-β-D-glucoside mixed micellar assay followed by a luminescence-based assay that measures the adenosine diphosphate (ADP) produced in a kinase reaction.28,29 DGKζ purified via size-exclusion chromatography was found to show strong kinase activity, and its specific activity was 3.6 nmol PA/min/μg. This value is comparable to those obtained from DGKα (type I isozyme) purified from porcine thymus (2.4 nmol PA/min/μg),34 DGKα from the baculovirus−insect cell expression system (2.0 nmol PA/min/ μg),27 and DGKε (type III isozyme) purified from the baculovirus−insect cell expression system (3.8 nmol PA/ min/μg).35 Moreover, no significant changes in the activity were observed after storage of the purified DGKζ at −80 °C for at least 1 month. We also determined the kinetic parameters of DGKζ for ATP and DG to assess its catalytic properties. An ATPdependent increase in the kinase activity was detected (Figure 2A), and the Km value was determined to be 0.050 ± 0.005 mM (Vmax: 4.05 ± 0.17 nmol PA/min/μg) (Table 1). The Km values for ATP obtained with DGKα purified from porcine thymus, expressed in COS-7 cells, and purified from the baculovirus−insect cell expression system were 0.1,34 0.24,27 and 0.1−0.25 mM,28,29 respectively. DGKε purified from bovine testis showed a Km value for ATP of 0.09−0.10 mM.36 We recently reported that the Km value for ATP of DGKη (type II isozyme) expressed in COS-7 cells was 0.052 mM.37 Taken together, the Km value of DGKζ (type IV isozyme) for ATP is lower than those of DGKα (type I isozyme) and DGKε (type III isozyme) and comparable to that of DGKη (type II isozyme).
successfully purified to near homogeneity as a monomer by a series of column chromatographies. The purified protein was fully active. Interestingly, the enzymatic parameters of DGKζ are different from those of other DGK isozymes.
■
MATERIALS AND METHODS Expression of DGKζ in Insect Cells Using a Baculovirus Expression Vector System. Expression of human full-length DGKζ (DGKζ-FL) and its catalytic domain (CD) alone (DGKζ-CD) was performed as previously described.27 The constructs of DGKζ-FL10 (aa 1−928 (UniProt accession ID: DGKZ Q13574-2)) or DGKζ-CD (aa 276−629) with an N-terminal His × 6 tag were amplified by polymerase chain reaction and inserted into the pOET3 vector (Oxford Expression Technologies, Oxford, U.K.) at SalI/NotI sites. Purification of DGKζ Expressed in Insect Cells. Purification of DGKζ-FL and DGKζ-CD using nickelnitrilotriacetic acid (Ni-NTA) agarose (Qiagen, Venlo, The Netherlands) and a Superdex 200 column 16/60 (GE Healthcare (Chicago, IL)) was performed as previously described.27 Collected fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) followed by Coomassie blue staining and Western blot analysis using the anti-DGKζ antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Protein quantification was performed by the Bradford assay or using the absorbance of a 1% solution of the protein in a 1 cm pathlength cell at 280 nm (E1% = 11.4). DGKζ Activity Assay in Vitro. The activity of DGKζ-FL was analyzed using the octyl-β-D-glucoside mixed micelle assay followed by the ADP-Glo kinase assay (Promega, Madison, WI) as previously described.28,29 To determine the kinetic constants, the activity assay was performed under a series of concentrations of adenosine triphosphate (ATP) (20−200 μm) and DG (0−5.4 mol %). For each reaction, 14.4 ng of purified DGKζ was added, and the assays were performed in triplicate for each ATP and DG concentration. The Km value was obtained by fitting the kinase activity of DGKζ with the Michaelis−Menten equation using Prism 5 (GraphPad Software, La Jolla, CA). Circular Dichroism Spectroscopy. Circular dichroism spectra were recorded under ambient conditions between 190 and 250 nm on a Jasco J-805 spectrometer (Jasco Corporation) using a cell with a path length of 0.2 mm, a scan speed of 60 nm/min, and a bandwidth of 1 nm. DGKζ for the spectrometry was prepared at 0.33 mg/mL (3.1 μm) in 20 mM Tris−HCl buffer, pH 7.4, 200 mM NaCl, 3 mM MgCl2, 0.5 mM dithiothreitol, and 5% glycerol. Five spectra were averaged and the spectrum obtained for the buffer was subtracted. Spectral data were analyzed using the program K2X30 suited in the DICHROWEB platform (http:// dichroweb.cryst.bbk.ac.uk/html/home.shtml).31 Statistical Analysis. Statistical comparisons were performed using a two-tailed t-test or a one-way analysis of variance followed by a Tukey’s test.
■
RESULTS
Expression and Purification of Full-Length DGKζ. It is difficult to express soluble and active DGK in bacterial expression systems. Therefore, we chose the baculovirus−Sf9 cell system to express DGKζ. Human DGKζ was reported to 5542
DOI: 10.1021/acsomega.9b00079 ACS Omega 2019, 4, 5540−5546
ACS Omega
Article
Figure 3. Effect of PS on purified DGKζ activity: purified DGKζ was incubated with various concentrations of PS as indicated for 5 min. Concentrations of the nonvaried assay components were 0.2 mM ATP and 5.4 mol % DG. Data are represented as the means ± SD for triplicate measurements.
DGKζ was reported to show no DG species selectivity.10,32 We analyzed the activities of purified DGKζ in the presence of several DG species, 12:0/12:0-, 16:0/16:0-, 16:0/18:1-, 18:1/ 18:1-, 18:0/18:0-, 18:0/20:4-, and 18:0/22:6-DG, as the substrate. When the activity with 12:0/12:0-DG was set to 100%, activities with 16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/ 18:0-, 18:0/20:4-, and 18:0/22:6-DG were all 78−108% (Figure 4). Thus, we confirmed that this isozyme does not have selectivity for a particular DG species (Figure 4).
Figure 2. Enzyme kinetics of purified DGKζ with ATP and DG. (A) ATP and (B) DG activity dependencies of the purified DGKζ as measured by the luminescence-based assay. DGKζ activity was plotted as a function of (A) ATP concentration (mM) or (B) DG concentration (mol %). Measured luminescence values were converted into the amount of ADP produced (nmol) based on the ATP-to-ADP conversion curve measured separately with a known concentration of ATP (50 μm to 1 mM). Data are represented as the mean ± SD for triplicate measurements.
Table 1. DGKζ Enzymatic Kinetic Parameters with ATP and DGa substrate
Km
Vmax
ATP DG
0.050 ± 0.005 mM 1.49 ± 0.12 mol %
4.05 ± 0.17 nmol/(min μg) 2.12 ± 0.06 nmol/(min μg)
a
Mixed micellar assay was performed to test the enzymatic activity over a series of substrate concentrations. The kinetic parameters were obtained as shown in Figure 2. Data shown are mean ± standard deviation (SD) for triplicate measurements.
Figure 4. Effects of various DG species on the activity of purified DGKζ. The purified DGKζ was incubated with 12:0/12:0-DG, 16:0/ 16:0-DG, 16:0/18:1-DG, 18:1/18:1-DG, 18:0/18:0-DG, 18:0/20:4DG, or 18:0/22:6-DG for 30 min. The concentrations of the assay components were 2.7 mol % DG, 0.2 mM ATP, and 13 mol % PS. Data are represented as the means ± SD for triplicate measurements. Statistical significance was determined by Student’s t test. Data shown are representative of three separate experiments.
The activity also increased in a DG concentration-dependent manner (Figure 2B), and the Km value was 1.49 ± 0.12 mol % (Vmax: 2.12 ± 0.06 nmol PA/min/μg) (Table 1). This value is comparable to those from our previous studies with DGKα purified from porcine thymus (3.3 mol %),34 from the baculovirus−insect cell expression system (1.1 mol %), and expressed in COS-7 cells (1.9−3.4 mol %).28,29 The Km value for DG of DGKε purified from bovine testis was 2.4 mM.36 On the other hand, the value of DGKη expressed in COS-7 cells was 0.14 mol %.37 Thus, the Km value of DGKζ for DG is comparable to those of both DGKα and DGKε and higher than that of DGKη. DGK is generally activated by an anionic phospholipid phosphatidylserine (PS).38 PS was reported to increase the activity of DGKζ in insect Sf21 cell lysates.39 Thus, we next examined the effect of PS on the activity of purified DGKζ (Figure 1). As shown in Figure 3, we confirmed that DGKζ activity increased in a PS-dependent manner. The ED50 value for PS was 8.6 mol % (Figure 3). The PS ED50 values for DGKα purified from porcine thymus and expressed in COS-7 cells were 1627 and 9 mol %,37 respectively. The ED50 value of DGKη for PS was 8.5 mol %.37 Therefore, The ED50 value of DGKζ for PS is comparable to those of both DGKα and DGKη.
Structural Characterization of the Purified DGKζ. We also revealed that the DGKζ solution could be concentrated using a centrifugal filter without any significant loss of the protein. Using the concentrated DGKζ (0.33 mg/mL (3.1 μm)), we attempted to understand the secondary structure composition using circular dichroism spectroscopy from 190 to 250 nm. The circular dichroism spectrum of DGKζ and following analysis using K2X30 suited in the DICHROWEB platform (http://dichroweb.cryst.bbk.ac.uk/html/home. shtml)31 indicates that DGKζ is well-folded and contains αhelical (25%) and β-strand (18%) structures (Figure 5A and Table 2), further demonstrating that the expression of a fulllength DGKζ in the baculovirus-infected insect cells is suitable for producing a natively folded and active form of DGKζ. The secondary structure composition of DGKζ was also assessed using the secondary structure prediction server self-optimized prediction method with alignment (SOPMA, https:// omictools.com/sopma-tool), which predicted the α-helix and β-sheet contents to be 30 and 18%, respectively (Figure 5B and Table 2). The values obtained from the experimental 5543
DOI: 10.1021/acsomega.9b00079 ACS Omega 2019, 4, 5540−5546
ACS Omega
Article
Using size-exclusion chromatography, DGKζ-FL eluted in a relatively sharp peak and remained as a monomer (Figure 1D,E). Such a production of DGKζ-FL in a soluble and monomeric form using the baculovirus−insect cell expression system is useful for the preparation of a DGKζ sample suitable for protein crystallization screening. DGKζ-FL was eluted at the molecular mass of 125 kDa. This value is larger than the calculated molecular mass (106 kDa), reflecting a multidomain architecture of DGKζ where each domain is connected by a flexible linker (Figure 1A). Spectral data analysis using the program K2X30 suited in the DICHROWEB platform31 suggested that the secondary structures of DGKζ are comprised of 25% α-helices and 18% β-strands, which are mostly in agreement with theoretical predictions (30% α-helices and 18% β-strands) (Figure 5 and Table 2). When compared with other mammalian DGK isozymes (DGKα and DGKε), the α-helical content of DGKζ is higher than that of DGKα (19%27) and lower than that of DGKε (29%35). The β-strand content of DGKζ is lower than those of both DGKα (27%27) and DGKε (22%35). DGKζ and DGKα have long regulatory regions in addition to the catalytic and C1 domains, whereas DGKε contains only C1 domains and the CD. Theoretical predictions (SOPMA) indicate that the C1 domains and CDs in DGKζ (C1 domains: 36%; CD: 36% (Figure 5)), DGKα (C1 domains: 22%; CD: 37%), and DGKε (C1 domains: 35%; CD: 32%) are relatively α-helixrich. Therefore, the α-helix content of DGKε is likely to be higher than those of DGKζ and DGKα. On the other hand, the ankyrin repeat is known to be α-helix-rich.40 DGKζ possesses four ankyrin repeats that contain 54% α-helices (Figure 5B). The ankyrin repeats would provide a higher α-helix content in DGKζ compared with DGKα. Enzymatic characterization of DGKζ, which was carried out for the first time, reveals that the Km values to ATP (0.050 mM) and DG (1.49 mol %) (Figure 2 and Table 1) are moderately lower than or comparable to those obtained using DGKα purified from porcine thymus,34 expressed in COS-7 cells,28,29 expressed in the baculovirus−insect cell expression system,27 and DGKε purified from bovine testis. Unlike DGKα and DGKε, DGKη has a high affinity for ATP (Km: 0.05 mM) and DG (Km: 0.14 mol %).37 In addition to different properties in DGK activities, the 1-MGK and 2-MGK activities of DGKζ are also different from those of other isozymes. Although type I (DGKα, β, and γ), II (DGKδ, η, and κ), and III (DGKε) isozymes have substantial 2-monoacylglycerol kinase (MGK) activity and the type V (DGKθ) isozyme possesses 1-MGK activity, DGKζ and ι (type IV) have only negligible 1-MGK and 2-MGK activities.41 Therefore, with regard to enzymatic properties, there are many kinds of DGK isozymes, suggesting that each DGK isozyme functions in different environments, which include distinct ATP and DG concentrations. Deletion of DGKα or DGKζ in mouse models results in T cells bearing a hyperresponsive phenotype through the attenuation of Ras guanyl nucleotide-releasing protein 1 and enhanced T-cell activity against malignancy.21,22,42,43 Therefore, both DGKα and DGKζ function as immunosuppressors in T cells. The functional similarities of DGKα and DGKζ suggest that they coordinate with and complement each other. Different enzymatic properties between DGKα and DGKζ would contribute to the cooperative and complementary actions of these isozymes in T cells. In summary, the present study demonstrates that the production of DGKζ-FL using the baculovirus−insect cell
Figure 5. Secondary structure of purified DGKζ: (A) Circular dichroism spectrum of DGKζ measured under ambient conditions between 190 and 250 nm on a Jasco J-805 spectrometer; DGKζ was prepared at 0.33 mg/mL (3.1 μm) in 20 mM Tris−HCl buffer, pH 7.4, 200 mM NaCl, 3 mM MgCl2, 0.5 mM dithiothreitol, and 5% glycerol. The analysis of the circular dichroism spectrum using the program K2X30 suited in the DICHROWEB platform31 showed the presence of both α-helical (25%) and β-strand (18%) structures. (B) Theoretical secondary structure analysis of DGKζ using SOPMA software.
Table 2. Secondary Structures of DGKζ secondary structure
DICHROWEB (%)
SOPMA (%)
α-helix β-strand
25 18
30 18
calculations and the theoretical predictions were mostly in agreement with each other.
■
DISCUSSION DGKζ is a lipid kinase that regulates a wide variety of cellular processes. Particularly, DGKζ has recently attracted much attention as a novel therapeutic target for cancer immunotherapy. However, no enzymatic or structural information on DGKζ is available, hindering our understanding of the catalytic mechanism of DGKζ and the strategy design of a reasonable structure-based drug development. In the present study, we purified a full-length form of DGKζ using the baculovirus− insect cell expression system. Moreover, we revealed its enzymatic and structural properties. In contrast to DGKζ-FL, the yield of DGKζ-CD was very low (Figure S1). 5544
DOI: 10.1021/acsomega.9b00079 ACS Omega 2019, 4, 5540−5546
ACS Omega
■
ABBREVIATIONS CD, catalytic domain; DG, diacylglycerol; DGK, diacylglycerol kinase; FL, full-length; PA, phosphatidic acid; PS, phosphatidylserine; WT, wild-type
expression system is a very useful approach to obtain DGKζ preparations for future functional and structural studies. First, DGKζ was purified to near homogeneity, and the purified DGKζ was soluble and monomeric, and was concentrated without any significant loss. These properties are essential for protein crystallization. Second, the obtained yield of DGKζ, 0.63 mg per 1 L cell culture, is sufficient to start crystal screening. Third, the purified DGKζ is catalytically sufficient. Fourth, the Km values for ATP and DG of DGKζ are different from those obtained for other DGK isozymes. Therefore, there are many enzymatically distinct DGK isozymes, suggesting that each DGK isozyme acts under different circumstances in cells. For example, DGKζ and DGKα, which act as physiological negative regulators of T-cell activation, are thought to function cooperatively and complementarily to each other. The successful purification of DGKζ in the present study permits detailed analyses of this important enzyme and will advance our understanding of DGKζ-related diseases and therapies.
■
■
REFERENCES
(1) Goto, K.; Nakano, T.; Hozumi, Y. Diacylglycerol kinase and animal models: the pathophysiological roles in the brain and heart. Adv. Enzyme Regul. 2006, 46, 192−202. (2) Mérida, I.; Avila-Flores, A.; Merino, E. Diacylglycerol kinases: at the hub of cell signalling. Biochem. J. 2008, 409, 1−18. (3) Sakane, F.; Imai, S.; Kai, M.; Yasuda, S.; Kanoh, H. Diacylglycerol kinases: why so many of them? Biochim. Biophys. Acta 2007, 1771, 793−806. (4) Topham, M. K.; Epand, R. M. Mammalian diacylglycerol kinases: molecular interactions and biological functions of selected isoforms. Biochim. Biophys. Acta 2009, 1790, 416−424. (5) Nishizuka, Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 1992, 258, 607−614. (6) Hurley, J. H.; Newton, A. C.; Parker, P. J.; Blumberg, P. M.; Nishizuka, Y. Taxonomy and function of C1 protein kinase C homology domains. Protein Sci. 1997, 6, 477−480. (7) Ron, D.; Kazanietz, M. G. New insights into the regulation of protein kinase C and novel phorbol ester receptors. FASEB J. 1999, 13, 1658−1676. (8) Exton, J. H. Phosphatidylcholine breakdown and signal transduction. Biochim. Biophys. Acta 1994, 1212, 26−42. (9) English, D. Phosphatidic acid: a lipid messenger involved in intracellular and extracellular signalling. Cell. Signaling 1996, 8, 341− 347. (10) Bunting, M.; Tang, W.; Zimmerman, G. A.; McIntyre, T. M.; Prescott, S. M. Molecular cloning and characterization of a novel human diacylglycerol kinase ζ. J. Biol. Chem. 1996, 271, 10230− 10236. (11) Topham, M. K.; Bunting, M.; Zimmerman, G. A.; McIntyre, T. M.; Blackshear, P. J.; Prescott, S. M. Protein kinase C regulates the nuclear localization of diacylglycerol kinase-ζ. Nature 1998, 394, 697−700. (12) Liu, Z.; Chang, G. Q.; Leibowitz, S. F. Diacylglycerol kinase ζ in hypothalamus interacts with long form leptin receptor. Relation to dietary fat and body weight regulation. J. Biol. Chem. 2001, 276, 5900−5907. (13) Yakubchyk, Y.; Abramovici, H.; Maillet, J. C.; Daher, E.; Obagi, C.; Parks, R. J.; Topham, M. K.; Gee, S. H. Regulation of neurite outgrowth in N1E-115 cells through PDZ-mediated recruitment of diacylglycerol kinase ζ. Mol. Cell Biol. 2005, 25, 7289−7302. (14) Mizuno, S.; Kado, S.; Goto, K.; Takahashi, D.; Sakane, F. Diacylglycerol kinase ζ generates dipalmitoyl-phosphatidic acid species during neuroblastoma cell differentiation. Biochem. Biophys. Rep. 2016, 8, 352−359. (15) Xiao, Y.; Peng, Y.; Wan, J.; Tang, G.; Chen, Y.; Tang, J.; Ye, W. C.; Ip, N. Y.; Shi, L. The atypical guanine nucleotide exchange factor Dock4 regulates neurite differentiation through modulation of Rac1 GTPase and actin dynamics. J. Biol. Chem. 2013, 288, 20034−20045. (16) Abramovici, H.; Mojtabaie, P.; Parks, R. J.; Zhong, X. P.; Koretzky, G. A.; Topham, M. K.; Gee, S. H. Diacylglycerol kinase ζ regulates actin cytoskeleton reorganization through dissociation of Rac1 from RhoGDI. Mol. Biol. Cell 2009, 20, 2049−2059. (17) Kim, K.; Yang, J.; Zhong, X. P.; Kim, M. H.; Kim, Y. S.; Lee, H. W.; Han, S.; Choi, J.; Han, K.; Seo, J.; Prescott, S. M.; Topham, M. K.; Bae, Y. C.; Koretzky, G.; Choi, S. Y.; Kim, E. Synaptic removal of diacylglycerol by DGKζ and PSD-95 regulates dendritic spine maintenance. EMBO J. 2009, 28, 1170−1179. (18) Arimoto, T.; Takeishi, Y.; Takahashi, H.; Shishido, T.; Niizeki, T.; Koyama, Y.; Shiga, R.; Nozaki, N.; Nakajima, O.; Nishimaru, K.; Abe, J.; Endoh, M.; Walsh, R. A.; Goto, K.; Kubota, I. Cardiac-specific overexpression of diacylglycerol kinase ζ prevents Gq protein-coupled
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00079. Expression of DGKζ-CD in baculovirus-infected insect cells and purification; schematic domain architectures; immunoblot and SDS-PAGE analyses (Figure S1) (PDF) Accession Codes
DGKZ Q13574-2.
■
Article
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel/Fax: +81-43-2903695. ORCID
Fumio Sakane: 0000-0003-0857-0377 Funding
This work was supported in part by grants from MEXT/JSPS (KAKENHI Grant Numbers: 26291017 (Grant-in-Aid for Scientific Research (B)), 15K14470 (Grant-in-Aid for Challenging Exploratory Research), and 17H03650 (Grantin-Aid for Scientific Research (B))); the Futaba Electronic Memorial Foundation; the Ono Medical Research Foundation; the Japan Foundation for Applied Enzymology; the Food Science Institute Foundation; the Skylark Food Science Institute; the Asahi Group Foundation; the Japan Milk Academic Alliance and the Japan Food Chemical Research Foundation (FS). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We would like to thank Dr Naoto Yonezawa (Chiba University) for giving us the Sf9 cells. We are grateful to Dr Takeshi Murata (Chiba University) for the use of their BioLogic chromatography system. 5545
DOI: 10.1021/acsomega.9b00079 ACS Omega 2019, 4, 5540−5546
ACS Omega
Article
receptor agonist-induced cardiac hypertrophy in transgenic mice. Circulation 2006, 113, 60−66. (19) Ali, H.; Nakano, T.; Saino-Saito, S.; Hozumi, Y.; Katagiri, Y.; Kamii, H.; Sato, S.; Kayama, T.; Kondo, H.; Goto, K. Selective translocation of diacylglycerol kinase ζ in hippocampal neurons under transient forebrain ischemia. Neurosci. Lett. 2004, 372, 190−195. (20) Saino-Saito, S.; Hozumi, Y.; Goto, K. Excitotoxicity by kainateinduced seizure causes diacylglycerol kinase ζ to shuttle from the nucleus to the cytoplasm in hippocampal neurons. Neurosci. Lett. 2011, 494, 185−189. (21) Olenchock, B. A.; Guo, R.; Carpenter, J. H.; Jordan, M.; Topham, M. K.; Koretzky, G. A.; Zhong, X. P. Disruption of diacylglycerol metabolism impairs the induction of T cell energy. Nat. Immunol. 2006, 7, 1174−1181. (22) Zhong, X. P.; Hainey, E. A.; Olenchock, B. A.; Jordan, M. S.; Maltzman, J. S.; Nichols, K. E.; Shen, H.; Koretzky, G. A. Enhanced T cell responses due to diacylglycerol kinase ζ deficiency. Nat. Immunol. 2003, 4, 882−890. (23) Chen, S. S.; Hu, Z.; Zhong, X. P. Diacylglycerol Kinases in T Cell Tolerance and Effector Function. Front. Cell Dev. Biol. 2016, 4, No. 130. (24) Mérida, I.; Torres-Ayuso, P.; Avila-Flores, A.; Arranz-Nicolas, J.; Andrada, E.; Tello-Lafoz, M.; Liebana, R.; Arcos, R. Diacylglycerol kinases in cancer. Adv Biol. Regul. 2017, 63, 22−31. (25) Riese, M. J.; Moon, E. K.; Johnson, B. D.; Albelda, S. M. Diacylglycerol Kinases (DGKs): Novel Targets for Improving T Cell Activity in Cancer. Front Cell Dev. Biol. 2016, 4, No. 108. (26) Singh, B. K.; Kambayashi, T. The Immunomodulatory Functions of Diacylglycerol Kinase ζ. Front Cell Dev. Biol. 2016, 4, No. 96. (27) Takahashi, D.; Sakane, F. Expression and purification of human diacylglycerol kinase alpha from baculovirus-infected insect cells for structural studies. PeerJ 2018, 6, No. e5449. (28) Liu, K.; Kunii, N.; Sakuma, M.; Yamaki, A.; Mizuno, S.; Sato, M.; Sakai, H.; Kado, S.; Kumagai, K.; Kojima, H.; Okabe, T.; Nagano, T.; Shirai, Y.; Sakane, F. A novel diacylglycerol kinase α-selective inhibitor, CU-3, induces cancer cell apoptosis and enhances immune response. J. Lipid Res. 2016, 57, 368−379. (29) Sato, M.; Liu, K.; Sasaki, S.; Kunii, N.; Sakai, H.; Mizuno, H.; Saga, H.; Sakane, F. Evaluations of the selectivities of the diacylglycerol kinase inhibitors R59022 and R59949 among diacylglycerol kinase isozymes using a new non-radioactive assay method. Pharmacology 2013, 92, 99−107. (30) Andrade, M. A.; Chacón, P.; Merelo, J. J.; Morán, F. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng. 1993, 6, 383−390. (31) Whitmore, L.; Wallace, B. A. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 2004, 32, W668−W673. (32) Ding, L.; Bunting, M.; Topham, M. K.; McIntyre, T. M.; Zimmerman, G. A.; Prescott, S. M. Alternative splicing of the human diacylglycerol kinase ζ gene in muscle. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 5519−5524. (33) Goto, K.; Kondo, H. A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11196−11201. (34) Sakane, F.; Yamada, K.; Imai, S.; Kanoh, H. Porcine 80-kDa diacylglycerol kinase is a calcium-binding and calcium/phospholipiddependent enzyme and undergoes calcium-dependent translocation. J. Biol. Chem. 1991, 266, 7096−7100. (35) Jennings, W.; Doshi, S.; Hota, P. K.; Prodeus, A.; Black, S.; Epand, R. M. Expression, Purification, and Properties of a Human Arachidonoyl-Specific Isoform of Diacylglycerol Kinase. Biochemistry 2017, 56, 1337−1347. (36) Walsh, J. P.; Suen, R.; Lemaitre, R. N.; Glomset, J. A. Arachidonoyl-diacylglycerol kinase from bovine testis. J. Biol. Chem. 1994, 269, 21155−21164.
(37) Komenoi, S.; Takemura, F.; Sakai, H.; Sakane, F. Diacylglycerol kinase eta1 is a high affinity isozyme for diacylglycerol. FEBS Lett. 2015, 589, 1272−1277. (38) Sakane, F.; Imai, S.; Kai, M.; Yasuda, S.; Kanoh, H. Diacylglycerol kinases as emerging potential drug targets for a variety of diseases. Curr. Drug Targets 2008, 9, 626−640. (39) Thirugnanam, S.; Topham, M. K.; Epand, R. M. Physiological Implications of the Contrasting Modulation of the Activities of the epsilon- and ζ-Isoforms of Diacylglycerol Kinase. Biochemistry 2001, 40, 10607−10613. (40) Li, J.; Mahajan, A.; Tsai, M. D. Ankyrin repeat: a unique motif mediating protein-protein interactions. Biochemistry 2006, 45, 15168−15178. (41) Sato, Y.; Murakami, C.; Yamaki, A.; Mizuno, S.; Sakai, H.; Sakane, F. Distinct 1-monoacylglycerol and 2-monoacylglycerol kinase activities of diacylglycerol kinase isozymes. Biochim. Biophys. Acta 2016, 1864, 1170−1176. (42) Jones, D. R.; Sanjuan, M. A.; Stone, J. C.; Merida, I. Expression of a catalytically inactive form of diacylglycerol kinase alpha induces sustained signaling through RasGRP. FASEB J. 2002, 16, 595−597. (43) Zha, Y.; Marks, R.; Ho, A. W.; Peterson, A. C.; Janardhan, S.; Brown, I.; Praveen, K.; Stang, S.; Stone, J. C.; Gajewski, T. F. T cell anergy is reversed by active Ras and is regulated by diacylglycerol kinase-α. Nat. Immunol. 2006, 7, 1166−1173.
5546
DOI: 10.1021/acsomega.9b00079 ACS Omega 2019, 4, 5540−5546
